This invention relates generally to a preparation of bone repair compositions, and more special refers to a novel technology in preparing collagen with inclusions of hydroxyapatite particles as bone repair compositions. This biocomposite material is designed for application as implant or growth stimulator of bone tissue in bone surgery, surgical stomatology, orthopedy, traumatology, reconstruction-restoration surgery and other fields of medicine.
One of the most important challenge, when producing a preparation which shall be used in reconstructive surgery, is to produce a substance with a chemical composition and parameters that are identical or nearly similar to the basic substance of the living organism. It has long been understood, that bone is composed of living cells, an organic matrix, and a mineral phase. About 95% of the organic matrix consists of the fibrous, matrix building protein collagen I, and the inorganic phase is mainly composed of calcium and phosphate in the form of crystalline hydroxyapatite. The mechanical properties of bone are governed by this perfect adapted composite structure. Whereas the mineral phase creates a high stiffness and compressive strength, the collagen matrix causes high fracture toughness.
The remodeling and repair processes in bone are strictly controlled by cellular activity of osteoblasts and osteoklasts. These cells are able to absorb and synthesize bone material to a limited extend. In order to produce bone-like implant material in medicine, especially in bone surgery, the bioceramic hydroxyapatite has found worldwide practical for the application at the human or mammalian body. Hydroxyapatite is one of the few bio-compatible mineral substances attracting a constant interest to its synthesis and properties. Different methods where developed for synthesizing HAP with different shape, porosity and size of crystals.
A known method of HAP production is described in the document PCT/IB 00/01232 where HAP particles with a length l=0.06 μm, width d=0.015 μm and thickness h corresponding to a crystal cell parameter equal to h=0.000688 μm or 0.000814 μm depending in the direction of the symmetry of the crystal cell are produced. The process is carried out in aqueous solution in three steps with a separation and concentration of suspension to produce paste or dry powder.
However, one disadvantage of this method is that the plate particles are produced with relatively large length and wide dimensions, i.e. having a relatively large size.
Therefore a wide range of materials has since been utilized, and elaborate designs have been disclosed for replacements of entire portions of bones, e.g., for hip joints (U.S. Pat. No. 3,820,167) and teeth (U.S. Pat. No. 4,186,486). Shaped and treated bone (U.S. Pat. No. 3,318,774), and various bone preparations such as bone dust compacted and flexible mats (U.S. Pat. No. 2,261,145) are used in as a bone-substitute, too. The aforementioned gold standard treatment, autogenous bone graft, with its good biological properties, adequate structural support and osteoinductiveness, has limitations and disadvantages because of the donor site morbidity, limited donor bone supply, anatomical and structural problems, high resorption levels during healing and high economical costs and hospitalization time. Other materials employed have included such as titanium (EPO Pub. No. 00712242), hydroxyapatite or ceramic such as aluminium oxide (U.S. Pat. No. 3,919,723). These materials however have drawbacks either, such as failure for complete bone ingrowth, extended inflammatory reaction and mechanical failure.
Among the potential substitutes another approach is mentioned in the paper “Biomimetric Mineralization of Collagen by Combined Fibril Assembly and Calcium Phosphate Formation” (Chem. Mater. 1999, 11, 2694-2701). There the synthesis of mineralized collagen is carried out by mixing of two solutions. First an aqueous solution containing CaCl2 and second an aqueous solution, actually being aqueous buffer solution containing potassium phosphate, NaCl and a HAP crystallization regulator. As a result the collagen fibrils mineralized with hydroxyapatite are produced with the HAP particle size to about 100 nm. The synthesis is carried out from 2 to 8 hours depending on the conditions. However, one of the main disadvantages of this known method is the production of relatively large size (100 nm) of hydroxyapatite particles and also the long synthesis time. Moreover, according to this method mineralized collagen is produced in narrow range of ratio HAP/collagen.
An object of the present invention is to provide a method to produce small hydroxyapatite particles effective and fast. Further object is to produce small hydroxyapatite particles in a matrix and to manufacture a novel implant material for bone surgery with a high biocompatibility.
The object is achieved by a method incorporating the features of Claim 1 wherein hydroxyapatite particles, in particular subnanodisperse hydroxyapatite particles are produced in a matrix in a method which comprises the steps of suspending a matrix building polymer in a solution forming a suspension, subjecting said suspension to a mechanical action and adding a solution of phosphoric acid and calcium hydroxide for synthesizing hydroxyapatite particles in the presence of the matrix building polymer. It turned out that a method comprising the steps of mixing and holding said suspension in a reactor thereby adjusting a defined alkaline pH value is advantageous. The amount of the matrix building polymer and aqueous solution of calcium hydroxide is controlled to achieve a required ratio of hydroxyapatite particles per matrix. As a result this composition comprises hydroxyapatite particles in the matrix, whereby in this porous sponge mass of composite both components were bond state by the adsorption forces on the molecular level or in a result of co-crystallization. The resulting biocomposite material is well suitable for medical treatments. Moreover said product comprises a very high uniformity and is cost effective, easy, fast and reproducible to manufacture.
According to the invention the matrix building polymer is a framework forming polymer in particular a fibrous protein, that encloses the hydroxyapatite particles. For this purpose proteins, polypeptides, fibrous biopolymers and any type of fibric proteins of collagen group are suitable matrix building polymers. Collagen may be replaced by gelatin or a comparable fibrous biomaterial to obtain a biocomposite material with similar properties and to gain a wider variety for the medical application of the above mentioned biocomposite material. Moreover the use of polysaccharides or derivates of polysaccharides as matrix building polymers is advantageous. In a preferred embodiment of the present invention the use of members of chitin like substances like chitosane is possible, too.
Furthermore the use of a mixture of the matrix building polymeric substances mentioned above, is possible.
According to the invention the present invention provides a novel manufacturing process for hydroxyapatite particles in a matrix, in particular a matrix of collagen, which produces a product with a high uniformity. According to the production of said biocomposite material it turned out, that it is favorable when the ratio of calcium hydroxide and phosphate in the suspension is 1.67±0.3 at the beginning of the hydroxyapatite particle synthesis. In view of temperature during synthesis of the hydroxyapatite particles a temperature between 1° C. and 45° C., preferred between 10° C. and 35° C. and particularly preferred between 20° C. to 25° C. is best suitable.
In the process it turned out, that it is favorable if the synthesis of the hydroxyapatite particles is carried out comprising the three steps of mixing and holding the suspension comprising the calciumhydroxide, the matrix building polymer and the phosphoric acid in a first mixing reactor (the first stage of mixing) with intensive stirring at Re≈104 thereby adjusting a defined alkaline first pH value of 8 to 9, feeding the suspension into a second reactor (the stage of complete substitution) for production of hydroxyapatite particles and causing a phase transformation of the suspension, and feeding the suspension into a third reactor (the second mixing stage) for mixing the suspension at a defined second pH value to then feed the mixture into a first reactor. Each of these steps is cyclically carried out in defined continuous periodical conditions within at least three stages thereby producing uniquely defined stoechiometric crystalline hydroxyapatite particles of chemically pure hydroxyapatite in a matrix.
As a result, by directing the production of hydroxyapatite particles and the matrix building polymer in the aforementioned predetermined crystallization route hydroxyapatite particles in the suspension are produced, such that the concentration of the fraction of the hydroxyapatite particles wherein more than 50%, 70% or preferably 90% of the particles are smaller than 3.5 nm. Preferably the fraction with a size from 0.35 nm to 5 nm is more than 50%, 70% or preferably 90% or the fraction from 0.35 nm to 2.5 nm is more than 50%, 70% or preferably 90%, or the fraction from 0.35 nm to 1.2 nm is more than 50%. In particular “smaller than” is to be understood as the particles having an overall size or length smaller than the given volumes or being within the given ranges, respectively.
According to the invention in general the synthesis of hydroxyapatite particles in the collagen matrix is carried out under different conditions by:    (a) Mixing a collagen solution in H3PO4 with Ca(OH)2 accompanied by co-crystallization of collagen and hydroxyapatite particles.    (b) Mixing a solution of collagen in a mixture of H3PO4 and HCl with a solution of Ca(OH)2 and NaOH to gain hydroxyapatite particles embedded in a matrix consisting of collagen and/or in the fibers.    (c) Mixing a two component solution comprising collagen in Na3PO4 and water with a solution of CaCl2 to gain a co-crystallization of collagen and hydroxyapatite particles.    (d) Mixing a collagen solution in H3PO4 with Ca(OH)2 as in method (c) and repeat mixing the product with the aqueous solution of collagen.
As a result a biocomposite comprising collagen and hydroxyapatite particles in different compositions is producable. This suspension, comprising the biocomposite material may be produced either as a paste with any ratio of hydroxyapatite particles/collagen or as a porous or as a powder mass. A matrix building polymer for example collagen of different compositions and concentrations mineralized with hydroxyapatite particles with the chemical formula is designed for application as implant material in bone surgery, orthopedics, traumatology, reconstruction-restoration surgery and other fields in medicine.
The present invention is described as follows in view of two preferred embodiments of methods for synthesis of the hydroxyapatite particles in a matrix of collagen.
The first preferred embodiment of the method according to the invention for producing the aforementioned subnanodisperse hydroxyapatite comprises the steps of intense mixing of an aqueous solution of calciumhydroxide and an aqueous solution of collagen in phosphoric acid in a first reactor, i.e. a mixing reactor. Preferably the formation of the hydroxyapatite particles takes place at a molecular ratio Ca/P=1.67.
In a preferred embodiment the mixing is carried out with the Reynolds number R≈104 by means of a blade mixer for about 15 min with a flow velocity between 10 m/s and 20 m/s. The ratio of hydroxyapatite particles/collagen is determined by the amounts of calcium hydroxide and collagen introduced into the reaction.
According to the invention the holding time after mixing is about 30 min. This holding time is advantageous to convert the primary composition of particles of calcium phosphates to hydroxyapatite particles according to the scheme DCP→OCP→TCP→HA (where DCP—dicalciumphosphate—CaHPO4, OCP—octacalciumphosphate (Ca8H2(PO4)6), TCP—tri calcium phosphate Ca3(PO4)2). The final pH value of the composition is 6 to 8.
According to this procedure a suspension of separate or particularly aggregated hydroxyapatite particles in collagen with sizes from 2 to 1000 nm, preferably 10 to 300 nm is produced where its bulk includes subnanoparticles (SNP) of HAP of given size.
Afterwards the separation and concentration by means of filtration or centrifugation or other methods to remove water and byproduct electrolytes, for example, by means of membrane technology at temperatures not exceeding 40° C.
In a result a paste is produced that presents a composition with a concentration of a sum of substances of collagen and hydroxyapatite particles from 5 to 30% by weight. During drying by means of lyophylic or sublimation drying processes a paste with any ratio of collagen/hydroxyapatite-water with concentration of a sum of substances of collagen and hydroxyapatite of more than 30% is produced. This paste may be used in medicine. Complete drying of the paste produces an anhydrous porous powder mass depending on the collagen content. This powder may be used in medicine, as well.
In a second preferred embodiment of the method according to the invention hydroxyapatite is produced in a method similar to process 1 to gain higher yields of subnanosized hydroxylapatite particles. This periodical process is carried out in three steps.
In a preferred embodiment the first reactor, i.e. a first mixing reactor is filled with aqueous solution of aqueous collagen phosphate solution and an aqueous suspension of calcium hydroxide taken in ratio Ca/P=1.67. The content of collagen and calcium hydroxide is regulated for producing the required ratio of HAP/collagen taking into account solubility of collagen in phosphoric acid.
According to the invention the holding time is shorter than 5 s, preferably about 0.2 s to 0.8 s, and the mixture is stirred with a flow velocity between 10 m/s to 20 m/s to achieve a complete homogenization of the medium components.
If the holding time is more than 0.8 s the formation of larger particles (up to 15-20 nm) is possible because aggregation of the primary subnanocrystals would proceed in the conditions of high hydrodynamics. At a holding time less than 0.2 s the formation of the primary subnanoparticle and their complexing with collagen is not complete.
At flow rates less than 10 m/s the required level of hydrodynamic conditions is not ensured and complete homogenization does not occur in the product whose size is not homogenized. A flow velocity of suspension larger than 20 m/s is not expedient because it results in increasing power consumption and in complicated technical construction of the first reactor. Though increasing flow rate during mixing does not affect the final product quality. The mixture is kept at a pH of 8 to 9.
At the second stage of the crystallization process that takes place in a reactor of complete substitution the conditions are created for modification of the chemical composition of the primary subnanoparticle with composition (CaHPO4)n through octacalcium phosphate and tri calcium phosphate to “Ca7(PO4)3OH-collagen” or “Ca10(Po4)OH)2 Collagen” at the extent of absorbing the Calciumhydroxide. The holding time of 10 s ensures completeness of the transformation of the primary subnanoparticle into hydroxyapatite. A holding time of more than 20 s is not expedient for the transformation process. A pH value in the range between 8 to 9 is optimal for the aforementioned transformation.
In a further preferred embodiment the produced mixture is led into a third stage that takes place in a second reactor of complete mixing. At this stage the saturation of the aqueous phase with calciumhydroxide takes place and pH attains about 12. In technological process the aqueous solution of calcium hydroxide is prepared during mixing the calcium oxide in water with a solid:liquid ratio (S:L) of 1:10 to 1:1000, to define the final concentration of the hydroxyapatite particles. The obtained aqueous mixture HAP-collagen and calcium hydroxide is led into the first stage to where collagen phosphate solution is fed. Then the mixture again is directed into the second and the third stages, whereby the realization of circulation of the whole reaction volume through three reactors is repeated cyclically until calcium hydroxide disappears, preferable completely and an aqueous suspension of HAP-collagen is formed with conservation of the aforementioned condition of the process each stage. The pH value after using calcium hydroxide in the whole volume of suspension is 6 to 8.
The circulation rate is determined by the quantities of reacting components, the holding time of reacting mass in the three stages and also by the indicated flow rates at these stages and it attains 10 to 15. The whole process is carried out at temperatures between 10° C. and 50° C. The characteristic feature of the crystallization of the composite “HAP-collagen” in the present invention is aggregation of collagen particles including hydroxyapatite particles. Their aggregation is proved to be inevitable due to mutual collisions in the intensive hydrodynamic conditions, multifold circulation of the reacting mass and high concentration of the composite particles HAP-collagen. High concentration is necessary for higher productivity if compared with the first method.
It should be noted that production of various ratios of HAP to collagen depends on the collagen solubility in phosphoric acid that limits its possibility of the wide variation of the composition in the final product. Collagen solubility depends on concentration of phosphoric acid and it is 1 to 20% and it is higher if the acid concentration increases. In a synthesis all phosphate ions should be introduced into hydroxyapatite. Therefore using such approach a product may be obtained containing less than 30% of collagen in HAP-collagen matrix if calculated for an anhydrous product. It is an advantage of the present invention that the synthesis results in the final product without any mixture of byproduct electrolyte.
To increase a ratio of HAP to collagen in the whole range, namely from the composition with 0.1% of collagen and 99.9% of HAP to one with 0.1% of HAP and 99.9% of collagen the compositions of the mentioned solution are varied and a mixture of hydrochloric acid and phosphoric acid is used to dissolve higher amounts of collagen. It relates to the fact that the solubility of collagen in hydrochloric acid is higher than in phosphoric acid in 2 to 5 times depending on the type of collagen. Therefore in the first preferred embodiment of the method of synthesis the following solutions are used:
1. Aqueous solution of CaCl2 and NaOH.
2. Aqueous solution of collagen in HCl and H3PO4.
By controlling the amount of a dissolved collagen and concentrations of HCl and H3PO4, and as well concentration of CaCl2 and NaOH (the ratio Ca/P is preferably equal to 1.67, and the final pH value is 6 to 8) it is possible to obtain any predetermined ratio HAP/collagen in a solid phase of the HAP-collagen product. Using for example, washing off or membrane technology it is possible to remove completely NaCl. Further separating and concentrating of the product are carried out to produce a paste with predetermined ratio HAP/collagen in the solid phase of the product, and by using the aforementioned means of drying the dry product is prepared with the same ratio of HAP/collagen. The production process itself is carried out under the same conditions as indicated in description of the first preferred embodiment of the method for producing hydroxyapatite particles in collagen.
For the second preferred embodiment of the method of synthesis the following solutions are used:    1. Solution of NaOH in aqueous suspension of calcium hydroxide.    2. Aqueous solution of collagen in HCl and H3PO4.
According to the invention this process is carried out under the aforementioned conditions for the second preferred embodiment of the method, adding the acidic solution of collagen to the solution of NaOH with calciumhydroxide. In this case by controlling the mass of collagen, H3PO4, HCl, calcium oxide and NaOH with molar ratio of about Ca/P=1.67 and complete neutralization of acid and alkalis (the final pH is 6 to 8) any required ratio HAP/collagen may be produced. In this case NaCl is formed in the produced mixture. Sodium chloride is removed from the system by means of the aforementioned methods. Further, concentrating the product suspension is carried out to produce the aqueous paste and/or dry product with any predetermined ratio of hydroxyapatite particles/collagen.
According to the invention a hydroxyapatite composition is obtained, comprising hydroxyapatite particles with dimensions from 0.35 nm to 3 nm, wherein the concentration of the fraction from 0.35 nm to 1.2 nm is more than 50% as shown in FIG. 3.
The aforementioned synthetic bone graft material accurately mimics the complex, multi-layered porous structure of a mammalian bone. The material and its particle size have been characterized to maximize the movement and attachment of bone cells within the implant. Recent clinical trials showed that nanocrystalline hydroxyapatite particles in a matrix of collagen results in rapid new bone growth, that is organized, well vascularized and of an extremely high stability.
In a preferred embodiment the bone-like implant material is produced in a different hydroxyapatite to collagen ratio to obtain a material suitable for a wide variety of medical applications.
In an advantageous embodiment said biocomposite material is ideal as bone implant material, dental filling material or dental cement. For this purposes the fraction of hydroxyapatite particles in the matrix of collagen should have a value between 30% and 90% by weight and particularly preferred between 40% and 70% by weight. For the application in the fields of wound healing or tissue engineering hydroxyapatite particle concentrations between 5% and 40% by weight, particularly between 5% and 20% by weight are suitable. In a preferred embodiment of the present invention said biocomposite material is part of a granulated powder, foil, mat, wound dressing, paste or wound healing formulation that is applied on a wound. Thus the migration and proliferation of cells involved in wound healing comprising vascular cells and fibroblasts is promoted and the osteoingrowth and differentation of osteoblasts is stimulated by subnanosized hydroxyapatite. Additional to the aforementioned applications said biocomposite material is suitable as a component of a mineralizing tooth paste or part of dry tablets for oral application for treatment of osteoporosis.
In an advantageous embodiment the implant material is producable in different shapes, thickness and with a variable content of water. The material is producable in form of a plaster of Paris that is biocompatible, bioactive and resorbable after 30-60 days. Moreover the biocomposite material formed as a putty, gel or injectable paste is a suitable filling material in the field of bone repair. In a preferred embodiment the bone-like tissue implant is produced in form of a coating that shows an enormous mechanical strength. It is possible to press or form the coatings to gain an implant material with a defined shape or caliper.
In an advantageous embodiment the bone-like tissue implant is an anhydrous powder which is optionally moulded in different sizes and shapes for various purpose. Preferably it is shaped in a mould which is preferably sealed. The plastic shape of the mould can be designed with computer aided medical image analysis technique which allows to replicate the missing bone structure. According to the invention it is possible to gain a biocompatible moulded pulp product with a variable size, morphology, porosity and geometry.
In a preferred embodiment of the present invention the bone-like implant can be used as a drug-delivery system.
This can be achieved generally by immersing the bone-like implant material in a solution of the desired cell growth factors or drug. Advantageously this results in an effective drug delivery mechanism to deliver high molecular weight proteins or drugs into the bone.
In accordance with the present invention this can be achieved. Thus high molecular weight proteins or drugs incorporated in the bone-like material e.g. in the pores of the subnanosized hydroxyapatit can be released from the bone graft to stimulate osteoingrowth, neovascularisation or to inhibit infections.
Thus growth factors include transforming growth factor (TGF-β), insulinelike growth factors (IGF)I and (IGF) II, plateled derived growth factors (PDGF), fibroblast growth factor (FGF), bone morphogenetic proteins (BMPs), RGD-Peptides, osteogenic protein (OP-1) and others; antibiotics, and other materials including vitamins, trace elements and minerals such as zinc can be incorporated into the bone-like implant material.
The following examples are given of the preferred embodiments of the methods according to the invention for producing subnanocrystalline hydroxyapatite in collagen matrix.